Continuing studies of pituitary tissues in the automated perifusion system confirm the observations of an age-related decrease in GnRH-stimulated LH secretion, previously shown in monolayer cultures. The failure to reverse the in vitro LH hyposecretion by repetitive perfusion pulses of GnRH parallels our prior observations after repetitive in vivo administration of GnRH and further suggests that aging is associated with an intrinsic pituitary gonadotropic secretary defect. Recent studies in primary monolayer cultures of heterogeneous pituitary cell suspensions reveal age-related decreases in secretion of LH after administration of the phorbol ester, PMA, a protein-kinase C activator as well as after nifedipine, a voltage gated calcium channel blocker. These observations suggest that both long and short term stimulation of LH release may proceed through separate mechanisms which are differentially affected by aging. Newer techniques for examining the cellular, biochemical, and molecular events responsible for age-related alterations in LH and PRL secretion have been introduced into our laboratory. These include; centrifugal elutriation, reverse hemolytic plaque assay, immunocytochemistry and immunofluorescence, RIA of G-protein subunits, and HPLC measurement of PIP metabolites. The effects of in vitro estrogen stimulation on LH-beta and PRL gene transcription is being studied in monolayer cultures of pituitary cells from old vs mature female rats.